Atrial fibrillation (AF), the most common sustained cardiac arrhythmia in adults, is associated with significant morbidity and increased mortality. While risk factors for AF are multifactorial, ~30% of patients have AF that is unassociated with underlying heart or systemic disease ('lone' AF). Over the last decade, we and others have shown that lone AF has a substantial genetic basis. The human cardiac sodium channel is responsible for the rapid upstroke of the cardiac action potential (AP) and blockers of Nav1.5, the canonical sodium channel encoded by SCN5A, are antiarrhythmic. We and others have shown that mutations in SCN5A cause a range of cardiac channelopathies including AF characterized by enhanced late sodium current (INa-L), prolonged AP duration and early afterdepolarizations. Genome wide association studies (GWAS) have implicated a second sodium channel Nav1.8, encoded by SCN10A, in AF susceptibility. We have identified multiple rare SCN10A variants in patients with lone AF. This raises the hypothesis, to be tested here, that rare variation in SCN10A is associated with AF. In Specific Aim 1, we will conduct a case-control genetic association study to determine whether a priori rare potentially pathogenic SCN10A variants are enriched in lone AF probands than in controls. In addition, we will ascertain extended pedigrees for each rare SCN10A variant carrier to determine familial aggregation with AF and determine genotype-phenotype correlations. Rare SCN10A variants that are highly conserved, predicted to be deleterious and aggregate with AF will be functionally characterized by expressing them in a dorsal root ganglia cell-line to determine the peak and INa-L. In vitro functional characterization of rare SCN10A variants will enable their AF-association and may provide novel insights into underlying genetic mechanisms of the arrhythmia. Furthermore, these studies also have the potential of uncovering Nav1.8 as a novel therapeutic target for antiarrhythmic intervention. In the previous cycle of this award, we used positional cloning and candidate gene approaches to identify genes encoding cardiac ion channels and signaling proteins which impart a large risk for the arrhythmia. In contrast, GWAS have identified common AF susceptibility loci with modest effects. While these approaches have provided important insights into the genetic architecture of AF, collectively they explain only a small fraction of the heritability of AF. This raises the hypothesis that rare variants with modest or large effects may be identified by whole exome sequencing (WES). In Specific Aim 2, we will screen a large cohort of patients with AF for rare variants in genes implicated by WES and GWAS as candidates for mediating AF susceptibility. We have identified four high priority novel candidate genes that cosegregated with AF using WES. Furthermore, in collaboration with other AF investigators, we have uncovered six additional AF susceptibility loci and five novel candidate genes when a meta-analysis of AF GWAS was performed. We will functionally characterize each gene variant using in vitro electrophysiology and in vivo zebrafish expression in order to determine their pathogenicity.